(A) Enpp2 and Ccl21 mRNA expression in LN stromal cells in Ccl19-Cre Enpp2fl/fl or Enpp2fl/fl mice. See also analysis of stromal cell populations in those mice in . (B) LPA-species distribution in the LNs of Ccl19-Cre Enpp2fl/fl and Enpp2fl/fl mice. Fluorescein-conjugated dextran (pseudocolored in yellow) was injected into the footpads to visualize the lymphatics, and draining LNs were collected 10 min after the injection. Signals corresponding to LPA (18:0) (ion transition from m/z 437 to 153), LPA (18:1) (from m/z 435 to 153), LPA (18:2) (from m/z 433 to 153), and LPA (20:4) (from m/z 457 to 153) were visualized by IMS analysis (magenta) and overlapped with MECA-79 staining (blue) on LN serial sections. LPA signals located within 50 μm of an HEV are circled by dotted lines and signals more than 50 μm away from an HEV are indicated by arrows for each of the LPA (18:1), LPA (18:2), and LPA (20:4) species. (C) The frequency of LPA signals by distance from an HEV, and the median distance from an HEV. Data are representative of three (A) or two (B, C) independent experiments. Differences between groups were evaluated by Student’s t-test. *P < 0.05, **P < 0.005, ***P < 0.0005. Bars, 100 μm.DOI: http://dx.doi.org/10.7554/eLife.10561.004

Intranodal T-cell migration in Enpp2fl/fl mice and Ccl19-Cre Enpp2fl/fl mice, analyzed by intravital two-photon microscopy. Intranodal T-cell migration in the PLN was analyzed 15–25 hr after injecting eGFP-expressing CD4+ T cells into Ccl19-Cre Enpp2fl/fl or Enpp2fl/fl mice. (A) Automated tracking of CD4+ T-cell migration (upper panels), and images rotated to display the z-dimension of the volume (lower). Trajectories of 50 randomly chosen cells are displayed as color-coded tracks to show movement over time, from blue (start of imaging) to red (end of imaging). (B) Translated tracking with a common origin. (C-E) Analysis of T cell motility. See also . (C) Average cell velocity. Each dot represents the average velocity of an individual cell, and bars indicate the median. Pooled data are shown in . (D) Mean displacement plot. (E) Motility coefficients, calculated from the slope of the regression line of the mean displacement plot as x2/6t, where x is the displacement at time t. Data represent the mean ± SD (C) or mean ± SEM (D, E). Data are representative of three independent experiments. Differences between groups were evaluated by Student’s t-test. *P < 0.05, **P < 0.005, ***P < 0.0005. Bars, 100 μm.DOI: http://dx.doi.org/10.7554/eLife.10561.007

WT CD4+ T cells were injected into Enpp2fl/fl or Ccl19-Cre Enpp2fl/fl mice, and their intranodal migration was analyzed by by two-photon microscopy. The data were pooled from two independent experiments. Differences between groups were evaluated by Student’s t-test. ***P < 0.0005.DOI: http://dx.doi.org/10.7554/eLife.10561.008

CD4+ T cells from WT or Lpar2-/- mice were left untreated or were treated with LPA (1 μM) and immediately loaded on one side of an EZ-Taxiscan chamber, and medium containing CCL21 (100 ng) was applied on the other side. (A) Cell migration on a surface coated with ICAM-1-Fc was monitored at 1-min intervals. (B) Cells that migrated toward the CCL21-containing contra-wells were counted, and the (C) average cell velocity and (D) turning angle were calculated. Bars represent the median. Differences between groups were evaluated by Student’s t-test. *P < 0.05, **P < 0.005.DOI: http://dx.doi.org/10.7554/eLife.10561.016

(A, B) The involvement of ROCK-myosin II signaling in LPA-enhanced cell migration across narrow pores. Lymphocytes were pretreated with the indicated concentrations of blebbistatin (Bleb), Y27632 (10 μM), or PTX (100 ng/ml), and were added with LPA to the upper chamber of a Transwell apparatus with a 3-μm-pore filter. After 2 hr, the migrated CD4+ T cells in the lower chambers were counted by flow cytometry. (C) Role of LPA2 in the LPA-induced chemokinesis of CD4+ T cells. WT or Lpar2-/- lymphocytes were added with various concentrations of LPA to the upper chamber. (D) Role of LPA2 in the LPA-induced activation of RhoA. WT and Lpar2-/- T cells were incubated with 1 μM LPA for the periods indicated, and were lysed immediately thereafter. RhoA-GTP in the lysates was pulled down using Rhotekin and was detected with an anti-RhoA antibody. (E) The relative band intensities of LPA-treated WT and Lpar2-/- T cells, normalized to untreated WT and Lpar2-/- T cells, respectively. (F) Effect of LPA on CD4+ T cell migration through Transwell membranes with a pore diameter of 3 or 5 μm. WT lymphocytes pretreated with or without LPA (1 μM) were applied to the upper chamber, and CCL21 (200 ng/ml) was added to the lower chamber. (G) Role of LPA2 in LPA-induced enhancement of CD4+ T cell chemotaxis toward CCL21. WT or Lpar2-/- lymphocytes were added with LPA (1 μM) to the upper chamber and CCL21 (200 ng/ml) was added to the lower chamber. Data are representative of two (A, B) or at least three (C–G) independent experiments (n = 3 per group). Data were evaluated by one-way ANOVA (A–C, F, G) or Student’s t-test (E) and represent the mean ± SD. *P < 0.05, **P < 0.005.DOI: http://dx.doi.org/10.7554/eLife.10561.017

(A) Illustration of the 3D migration assay. The cell suspension was mixed with collagen gel (1.6 mg/ml). CCL21 (5 μg/ml) was loaded into one side of the gel, and LPA (5 μM) was applied to both sides. The migration of eGFP-expressing CD4+ T cells or their Lpar2-/- counterparts was recorded for 120 min by time-lapse microscopy. (B) Manual tracking of T cell motility. (C) T cell velocity. See also the directionality of T cell movement in . (D) Involvement of myosin II/ ROCK in LPA-induced T cell motility. After eGFP-expressing CD4+ T cells and WT counterparts were treated with vehicle and indicated inhibitors, respectively, they were mixed with the collagen gel and monitored for cell migration. Data are representative of two independent experiments (B, C) and pooled from two independent experiments (D). Differences between groups were evaluated by Student’s t-test (C) or one-way ANOVA (D). *P < 0.05, **P < 0.005, ***P < 0.0005.DOI: http://dx.doi.org/10.7554/eLife.10561.019

Effect of LPA on the directionality of T-cell movement in 3D collagen gel.

WT and Lpar2-/- CD4+ T cells were mixed with collagen gel (1.6 mg/ml). After applying CCL21 into one side of the gel, the T-cell migration in the collagen gel in the presence or absence of LPA was monitored by time-lapse microscopy. Directionality of cell migration was calculated as (net displacement)/(total path length).DOI: http://dx.doi.org/10.7554/eLife.10561.020